Comparative analysis of soil nematode biodiversity from five different fruit orchards in Osmaneli district, Bilecik, Türkiye

Abstract Nematode population densities were determined in 60 soil and root samples collected from 6 fruit orchards in the Bilecik province (western Turkey), between April 2022 and June 2022. The total number of identified nematodes have reached up to 2418 individuals (number of female: 1036; male: 154; and juvenile: 1228). They belong to 54 species, 54 genera, 33 families and 11 orders. Plant parasitic nematodes that were detected mostly are listed as follows: Helicotylenchus (6,12 %), Pratylenchus (5,74 %), Paratylenchus (4.83 %), Xiphinema (3,06 %), Tylenchorhynchus (2,19 %), Malenchus (1.94 %) and Tylenchus (1.19 %). According to the maturity index analysis, mean values showed the highest maturity level at peach trees (MI value: 3,52), followed by; walnut trees (MI value: 2.49), cherry trees (MI value: 2.15), nectarine trees (MI value: 1.86), plum trees (MI value: 1.57), and olive trees (MI value: 1.42). Mostly the diverse group in terms of species richness was within the order Dorylaimida. The nematodes associated with peach and walnut trees here showed the most stable environments in terms of soil nematode community structure.


Introduction
Turkey is in an advantageous position in fruit growing thanks to its location and ecological characteristics.It is known that there are many species and varieties richness in the country due to the fact that it is located in the Near East and Mediterranean gene centers (Erdem and Kos, 2022).The country is the homeland of many fruit species such as that of the apple (Malus domestica L.), pear (Pyrus communis L.), quince (Cydonia oblonga L.), hazelnut (Corylus avellana L.), pistachio (Pistacia vera L.), sour cherry (Prunus cerasus L.), cherry (Prunus avium L.), plum (Prunus domestica L.), walnut (Juglans regia L.), almond (Prunus dulcis L.), chestnut (Castanea sativa L.), fig (Ficus carica L.), grape (Vitis vinifera L.) and pomegranate (Malum granatum L.), which have an important place in fruit culture.In 2020, a total of 21,853,084 tons of production were realized with an increase of approximately 6% compared to the data of 2019 (Gerçekçioğlu et al., 2009).
The Bilecik Province is located in the southeast of the Marmara Region, between the intersection points of the Marmara, Aegean, Central Anatolia, and Black Sea regions.The region has many advantages in fruit and vegetable cultivation thanks to its location, changing altitude differences in its geography and the unique ecosystem that is surrounded by the Sakarya River.The cultivation of many fruits is wellknown locally, such as peach in the Osmaneli district, pomegranate cultivation in the Inhisar district and walnut and cherry cultivation in the Gölpazarı district.Furthermore, Bilecik is at the midpoint of cities such as Istanbul, Bursa, Izmit and Eskisehir, and it also provides access to the Marmara, Central Anatolia and Mediterranean Regions by road and railway, thus providing easy access to airports and ports, and therefore to the market (Niyaz and Demirbaş, 2011) (Table 1).
Nematodes (Phylum Nematoda) are one of the most diverse groups of invertebrate animals, characterized by a simple body structure with wide variety of feeding habits, life strategies, and their important role in the soil food web.It is reported that nearly 25.000 nominal species have been identified (Zhang, 2012), and it is estimated that their diversity can reach up to 1.000.000nematode species (Hugot et al., 2001).
As with any other crop grown in Türkiye, damage to the tree and/or fruit by pests and diseases, including nematodes, reduces the grower's profit.The effects of some plant parasitic nematodes on plant growth and yield are largely the result of the disruption that these organisms cause to the normal process of plant root growth and soil exploration for both water and nutrients.Nutrient deficiencies resulting from the failure of the plant root system to explore and exploit the soil adequately can also be a major consequence of a plant parasitic nematode attack.
The objectives of this study were, (i) to determine the soil nematode fauna of fruit orchards in Osmaneli District of Bilecik Province, (ii) to characterize of nematodes as soil bioindicators and (iii) to characterize the biodiversity of nematodes in regard to their host plant.

Sampling
Samples were collected at the Osmaneli district, Bilecik, Turkey, in April 2022 during a field survey.The sampling was done regarding fruit tree orchards soil habitats and along six different eco-habitats, namely: cherry, nectarine, olive, plum, walnut and peach trees.(Fig. 1; Table 2).
Samples were collected from 60 sampling sites (10 samples from each fruit type).For each location, one soil sample was collected from a 15 x 15 cm plot.A total number of 60 soil samples were put into ziplock sampling bags, stored in portable cooler during transportation and brought to the nematology laboratory of Duzce University for the extraction process.

Extraction
A modified Baermann's (1917) funnel technique using 12 cm diameter petri dishes was used during the extraction of nematodes.After separating rocks, 100 g of fresh soil was evaluated from each sampling site.Plastic trays lined with paper towels were used for extraction and incubated for 48 hours in the nematology laboratory.Extracted nematodes were collected after 48 hours.Nematode suspensions were heated up to 60 °C for killing before fixation.
A formalin solution of 4% was used for fixation and preservation of nematodes until preparing permanent glass slides.Extractions were labeled with the relevant sample number, transferred to plastic tubes, and stored at Düzce University Nematology Laboratory.
The rest of the soil samples were also stored in the soil laboratory for having a backup requirement in case of future studies.

Recovery of entomopathogenic nematodes
A 100 g soil sample from each sampling site was placed into a glass container each with three last instar larvae of the wax Moth Galleria mellonella (L.) and covered with a lid.(Bedding and Akhurst, 1975;Kaya and Stock, 1997).The samples were then stored at room temperature.After 10 days, dead larvae were collected and transferred to White traps to collect the emerging IJs (Kaya and Stock, 1997).

Preparation of Nematodes for Light Microscope
After picking up procedure, preserved nematodes were rinsed with purified water to remove the debris.
A staining block of 1.25 cm deep which contained 96% ethanol with the extracted nematodes was placed in an incubator at 40 °C, and a few drops of glycerol: formalin (4 %) (1:99) were added and left at room temperature overnight.The next morning, a few drops of a solution of five parts glycerol and 95 parts of 96 % ethanol were added, and two-thirds of its cavity was covered with a glass square.A few drops of the glycerol: ethanol (5:95) solution were added every two hours for the gradual transition of the glycerin.At the end of the day, two drops of glycerol: ethanol (50:50) were added to the staining block.The next day, individual nematodes were covered with glycerol and permanent glass slides were prepared (Yoder et al., 2006).

Nematode Identification and analysis of ecological parameters
Nematodes were identified manually by using an Olympus CH microscope (Olympus Optical, Tokyo, Japan).Classification of nematodes were determined

Results
The total number of identified nematodes reached up to 2418 individuals (number of female: 1036; male: 154; and juvenile: 1228) belonging to 54 species, 54 genera, 33 families and 11 orders (Table 3).Besides, the total nematode abundance showed variability among samples with an average number of nematodes per 100 gr of soil that were 2 to 145 individuals from the sampling sites (Table 2).

Maturity Index Analysis
According to the maturity index analysis (Fig. 2; 3), mean values showed the highest maturity level at peach trees (MI value: 3,52), followed by; walnut trees (MI value:  Food web analysis of soil properties provides a useful tool for predicting soil quality by enrichment and structure type parameters.Results showed that the two of the fruit orchards, nectarine and plum, nematode assemblage were yielded into a high enrichment class, which means disturbed, N. enriched, with low C:N value, high bacterial activity and conductive soil.Three of the fruit trees' (Olive, walnut and peach) nematode assemblage were placed into maturing, N-enriched, with low C:N value, high bacterial activity, and regulated class.Nematode JOURNAL OF NEMATOLOGY assemblage of the cherry tree orchard occurred at degraded, depleted, with high C:N value, more fungal activity and a conductive soil type (Fig. 4).

Faunistics
The Bilecik Province, located in the Marmara Region of Turkey, is a region where agricultural production is intense.In this sense, when we look at the structure of the fruit trees in the region, it is possible to say that stone fruits are generally grown.However, fruits such as olives and walnuts are widely grown in the region.In the specific Osmaneli district of the Bilecik province, no holistic study on soil nematode diversity was found in this region in the literature research.However, this climate zone, which is fed with rich water resources with its proximity to the Sakarya river, is a region rich in terms of highly fertile soil structure and therefore biodiversity.Among the studies, records from Turkey were presented in the study of plant parasitic nematode fauna in olive trees in the Mediterranean climate zone (Ali et al., 2014).When the studies on soil nematode fauna from the Marmara Region (Öztürk, 2023) were examined, it was determined that there were similar taxa.Likewise, a large-scale survey study was conducted mainly on entomopathogenic nematodes (Güneş and Gozel, 2011) from orchards in the Marmara Region.
The rising interest of learning about nematode community behavior in the soil food web is allowing us to monitor their role in the ecosystem.In Türkiye, the discovery of soil nematodes and especially the diversity of free-living nematodes are still receiving very little interest from the scientific community.The aim of this study is to determine soil nematode fauna in the five different fruit orchards in Bilecik Province, Marmara Region with a holistic approach and to identify vermiform terrestrial nematodes up to the genus level.To date, Anatonchus Cobb, 1916, Diphtherophora de Man, 1880, Funaria Linde, 1938, Telotylenchus Siddiqi, 1960and Tripylella Brzeski and Winiszewska-Slipinska, 1993 were not reported in any scientific report from Turkiye and apparently, this study is the first report of these genera regarding Turkiye's nematofauna.Our contribution therefore is  Nematodes as biological indicators provide valuable information about soil health.The study is conducted in five different fruit orchards habitats namely that of cherry, nectarine, olive, peach, plum and walnut.On the other hand, fruit tree orchards and their ecosystem are rarely discovered in terms of terrestrial nematofauna in Türkiye.Unfortunately, there is no study to our knowledge, related to freeliving nematode biodiversity in the Marmara region.Regarding the total diversity, this study indicates a valuable contribution on the importance of faunistic studies of terrestrial nematode species.

Species distribution and nematode abundance
The distribution of species in the fruit trees' rhizosphere was investigated with an integrative approach to the species relative abundance and occurrence patterns.In fact, it is a difficult task to interpret the results of the distribution of soil nematodes.However, here we applied an interpretation scheme that consists of three frequency classes (low, medium, and high frequency) in terms of distribution patterns of nematodes in five different fruit types.According to these results, nematode genera that occurred in a high frequency at all fruit tree orchards are listed as follows: Acrobeloides, Chiloplacus, Eucephalobus, Helicotylenchus, Malenchus, Panagrolaimus, Pratylenchus and Tylenchorhynchus.These are cosmopolite nematode species and are distributed all over the world.Nematode genera that are occurring in a medium frequency at all fruit tree orchards are listed as follows: Aphelenchoides, Aphelenchus, Aporcelaimellus, Paratylenchus, Tylenchus, and Xiphinema.Finally, nematode genera that are occurring in a low frequency are listed as follows: Alaimus, Amplimerlinus, Anatonchus, Aporcelaimus, Basiria, Belondira, Belonolaimus, Cephalobus, Cervidellus, Chromadorea, Clarcus, Criconema, Diplogaster, Dipterophera, Ecumenicus, Eudorylaimus, Eumonhystera, Filenchus, Funaria, Geomonhystera, Heterodera, Hoplolaimus, Labronema, Microdorylaimus, Monhystera, Mononchus, Mylonchulus, Nagelus, Nygolaimus, Plectus, Prionchulus, Prismatolaimus, Rhabditis, Rotylenchus, Telotylenchus, Teratocephalobus, Tobrilus, Tripylella, Tylocephalus and Steinernema.Here, we need to place particular focus on the entomopathogenic nematode Steinernema spp., which occurred only in Plum and Olive orchards.Entomopathogenic nematodes are obligate parasites on insects in the soil and have a high potential to suppress many important pests, especially on fruit orchards, with the ability to survive for a long time under suitable conditions (Bedding et al., 1983;Kaya, 1985;Bedding, 1990).

The nematode community
Comparing the nematode trophic groups within five fruit tree types showed a similar pattern.However, there are small differences that can be seen as a result of fruit tree characteristics.Apparently, bacterivorous nematodes were found to be the most common group in all the samples except in cherry trees.Plant parasitic nematodes were the most abundant trophic group in cherry orchards (69.1 %).Fungivorous nematodes have reached the highest percentage at nectarine orchards (16.7 %).On the other hand, in olive tree orchards, bacterivorous nematodes have reached up to 70.7 % of the total nematode community.Predator nematodes were found the most in plum tree orchards (47.3 %) as well as omnivore nematodes (6.8 %).
Additionally, the most diverse group in terms of species richness was within the order Dorylaimida.Omnivore nematodes as a persistent group in terms of soil monitoring, were found only in peach (6.8 %) and walnut (1.9 %) tree orchards with low percentages.The persistence of this group stems from their biology which also refers to an occurrence in mature and fertile soils and having a long-life cycle.The total percentage of omnivorous and predator nematodes has reached 54.1 % of peach tree orchards which shows clues of soil maturity and an undisturbed ecosystem in these areas.Walnut tree nematode assemblage was the most equalized composition in terms of feeding types.On the other hand, the abundance of bacterivorous, fungivorous, and plant parasitic nematodes had little variation between the sampling sites.This shows clues about the disturbance and agricultural practices' pressure on the nematode community structure.
The balance of an ecosystem can be approached by the composition of nematodes in the soil.According to the colonizer-persister (cp) structure of the nematodes in these five fruit orchards, it is possible to say the nematodes associated with peach and walnut trees here showed the most stable environments in terms of soil nematode community structure.Schnürer et al. (1986) andYeates (2007) mentioned that the most important factors affecting the nematode community are the environmental effects of regional and seasonal changes such as soil organic matter, texture, structure, chemical

Figure 2 :
Figure 2: Comparative maturity index analysis of nematode c-p classification from five fruit orchards in Osmaneli, Bilecik, Türkiye.

Figure 5 :
Figure 5: Feeding types and their relative abundance of nematodes at different fruit tree orchards.

Table 1 .
Bilecik Province fruit production area and production amount of selected fruit types in 2022.* *According to(Erdem & Kos, 2022)

Table 2 .
GPS Coordinates of the sampling sites; the location and host plant association.
of the nematode community composition in the ecosystem.The Nematode Indicator Joint Analysis calculation system(Sieriebriennikov et al., 2014)was used to analyze food web structure, feeding type diagnostics and MI family indices.